Antibodies to cloned Leptospira outer membrane protein and uses therefore

ABSTRACT

An antigenic preparation is provided which contains a 63 Kd outer membrane protein from Leptospira which can be used immunologically as a vaccine for leptospirosis caused by this organism. Also provided in the invention are polynucleotides encoding the protein and antibodies which bind the protein which are useful in the diagnosis and treatment of leptospirosis.

This is a divisional of U.S. application Ser. No. 08/886,863, filed Jul.1, 1997, issuing Oct. 20, 1998 as U.S. Pat. No. 5,824,321, which is adivisional application of U.S. patent application Ser. No. 08/249,013,filed May 25, 1994, issued on Jul. 1, 1997 as U.S. Pat. No. 5,643,754.

This invention was made with Government support by the Veteran'sAdministration Research Advisory Group and Grant Nos. Al-21352,Al-29733, and Al-12601 awarded by the National Institutes of Health. TheGovernment has certain rights in the invention.

BACKGROUND OF THE INVENTION

1. Field of the Invention

This invention relates generally to an antigenic preparation andspecifically to a Leptospira outer membrane protein (OmpL2) which isused to induce a protective immune response in animals. Such a proteincan be used immunologically as a vaccine for leptospirosis caused bythis organism. Alternatively, diagnosis of leptospirosis can beperformed by detecting the presence of the protein, antibody to theprotein, or polynucleotide which encodes the protein.

2. Description of Related Art

Leptospirosis is a widespread zoonotic disease caused by pathogenicstrains of Leptospira which are capable of infecting most mammalianspecies. At present, there are six pathogenic species and threenonpathogenic species within the genus Leptospira. Infection occurseither through direct contact with an infected animal or indirectcontact with contaminated soil or water. In livestock, the diseasecauses economic losses due to abortion, stillbirth, infertility,decreased milk production, and death.

Efforts to control leptospirosis have been hampered because virulentleptospires have the capacity for both long-term survival in theenvironment as well as persistent infection and shedding by wildlife andlivestock. Currently available leptospiral vaccines produce short-termimmunity and do not provide cross-protection against many of the 170serovars of pathogenic Leptospira (Thiermann, et al., J. Am. Vet. Med.Assoc. 184:722, 1984). These vaccines consist of inactivated wholeorganisms or outer envelope preparations which produce seroreactivity asdetermined by microscopic agglutination of intact organisms. The natureof the protective immunogens in these vaccine preparations has not beenconclusively elucidated, although several lines of evidence suggest thatlipopolysaccharide-like substance (LLS) may confer a degree ofprotection.

The pathhogeanesis of leptospircsls is veny similar to that of otherspirochetal diseases, including syphillis (caused by Treponema pallidum)and Lyme borreliosis (caused by Borrelia burgdorferi). Both syphilis andLyme borreliosis are characterized by widespread dissemination early inthe course of disease, including invasion of the central nervous system.Leptospira share this ability with other pathogenic spirochetes suchthat meningitis is a common manifeslation of leptospirosis. Anotherfeature of spirochetal infections is the ability to persist chronicallyin the host, as manifested in cases of tertiary syphilis and chronicLyme arthritis.

In attempting to identify leptospiral outer membrane proteins (OMPs),previous research was unsuccessful due to such problems as: 1) thetechniques used to identify surface-exposed proteins probably involveddamage to the fragile leptospiral outer membrane resulting in exposureof subsurface structures; 2) putative surface-exposed proteins that wereidentified included a 35-36 kD doublet corresponding to Leptospiraendoflagella (Kelson, et al., J. Med. Microbiol. 26:47, 1988), which aresubsurface structures in spirochetes; and 3) use of SDS whichnonselectively solubilizes proteins irrespective of their nativecellular location.

Nunes-Edwards, et al. (Infect. Immun. 48:492, 1985) introduced the useof radioimmunoprecipitation and cell fractionation schemes based on theuse of SDS in an effort to identify leptospiral OMPs. The leptospiresused in their radioimmunoprecipitation procedure were subjected to highspeed centrifugation (20,000×g) prior to the addition of antibody. Suchhigh centrifugal forces cause mechanical disruption of the leptospiralouter membrane. Niikura, et al. (Zbl. Bakt. Hyg. A. 266:453, 1987)immunoprecipitated SDS-solubilized extracts of virulent and avirulentstrains of L. interrogans serovar copenhageni that had been labeled bylactoperoxidase-catalyzed surface radioiodination. Since both of thesestudies precipitated a 35-36 kD doublet consistent with leptospiralendoflagella, there was a concern as to whsther the other prot-insidentified might also have a subsurface rather than a surface location.

Jost, et al. (J. Med. Microbiol. 27:143) characterized a monoclonalantibody with specificity for a 35 kD proteinase K sensitive antigenwhich was present in a leptospiral outer envelope preparation. However,to demonstrate binding of the monoclonal antibody by immunoelectronmicroscopy, the leptospiral outer membrane had to be disrupted. Doherty,et al. (J. Med. Microbiol. 28:143) cloned two leptospiral proteinsrepresented in an SDS-generated outer membrane preparation of L.interrogans, but did not provide corroborating evidence that theseproteins are either constituents of the outer membrane or aresurface-exposed.

Unsuccessful research on the identification of Leptospira and T.pallidum OMPs has shown the importance of taking into accountspirochetal outer membrane fragility and the lack of outer membraneselectivity of ionic detergents such as sodium dodecyl sulfate (SDS)(Cunningham, et al., J. Bacteriol. 170:5789, 1988; Penn, et al., J. Gen.Microbiol. 131:2349, 1985; Stamm, et al., Infect. Immun. 55:2255, 1987).Outer membrane proteins are of great importance because they play a keyrole in bacterial pathogenesis. The identification of outer membraneproteins involved in Leptospira pathogenesis is significant tounderstanding not only leptospiral outer membrane proteins and theirinvolvement in pathogenesis, but also to understanding other spirochetalouter membrane proteins and their role in pathogenesis.

SUMMARY OF THE INVENTION

The present invention is based on the identification of OmpL2 as aleptospiral outer membrane protein which is associated with pathogenicstrains of Leptospira. Due to spirochetal outer membrane fragility andthe fact that outer membrane proteins are present in small amounts,there have been no definitive reports of membrane spanning spirochetalouter membrane proteins until the present invention. The inventiondescribes a 63 kD outer membrane protein from Leptospira and the geneencoding the protein. The deduced amino acid sequence has a typicalleader peptidase I cleavage site, implying export beyond the innermembrane. The 63 kD protein has been designated OmpL2 for outer membraneprotein of Leptospira. This immunogenic polypeptide is useful forinducing an immune response to pathogenic Leptospira as well asproviding a dianostic target for leptospirosis.

BRIEF DESCRIPTION OF THE DRAWINGS

FIGS. 1A, 1B and 1C show the DNA sequence and deduced amino acidsequence of OmpL2.

FIG. 2 shows an amino acid comparison between OmpL2 and eightTonB-dependent outer membrane proteins for seven regions of homologyidentified by Kadner, R., (Molecular Microbiology, 4:2027, 1990).

FIG. 3 shows a topological model of OmpL2. Membrane spanning beta-sheetsare shown within rectangles in a staggered array with the hydrophobic,membrane-facing residues on the right side of the array.

DETAILED DESCRIPTION OF THE INVENTION

The present invention provides an isolated immunogenic polypeptide froman outer membrane protein of a pathogenic Leptospira species. Alsoincluded is a polynucleotide sequence which encodes the polypeptide. Theouter membrane protein is a 63 kD protein originally isolated fromLeptospira alstoni which has been termed OmpL2 and is apathogen-associated exported protein of Leptospira. This immunogenicpolypeptide is useful in a pharmaceutical composition for inducing animmune response to pathogenic Leptospira.

The invention includes a method of producing the polypeptide portion ofan outer membrane protein of Leptospira using recombinant DNAtechniques. The gene for the L. alstoni OmpL2 outer membrane protein iscloned into a plasmid vector which is then used to transform E. coli.When the OmpL2 gene is expressed in E. coli, the polypeptide producedhas a molecular weight of approximately 63 kD as determined bySDS-polyacrylamide gel electrophoresis.

Recently, one approach to studying genes encoding exported leptospiralproteins was developed based on the concept underlying TnphoAtransposition (Boquet, et al., J. Bacteriol. 169:1663, 1987; Hoffman, etal., Proc. Natl. Acad. Sci. USA, 82:5107, 1985; Manoil, et al., Science233:1403, 1986; Manoil, et al., J. Bacteriol. 172:515, 1990). The systemutilizes a phoA expression vector termed pMG, that contains an alkalinephosphatase (AP) gene lacking its signal sequence, together with the E.coli mutant strain KS330 (Strauch, et al., Proc. Natl. Acad. Sci., USA85:1575, 1988), which possesses a leaky outer membrane, to identifygenes encoding signal peptide export-dependent proteins which mayfunction as virulence determinants. The screen for genes which encodeexported proteins is done by identifying blue-halo colonies. The utilityof this system has been confirmed for both Treponema pallidum (Blanco,et al., Mol. Microbiol. 5:2405, 1991) and Leptospira alstoni in whichsignal peptide containing proteins from both organisms were shown to beexported in E.coli. Such a method was utilized for identification of theompL2 gene of the invention.

Sequence analysis showed that the OmpL2 structural gene consists of 1740bases encoding a protein of 540 amino acids (SEQ ID NO:1 and 2). Asexpected for proteins to be exported beyond the inner membrane, thederived amino acid sequence begins with a 24-residue signal peptide. TheOmpL2 sequence contains 24 stretches of amphipathic beta-sheetstructure, consistent with outer membrane protein transmembranesegments, making it possible to propose a topological model with largesurface-exposed loops and short periplasmic loops typical of outermembrane proteins.

Comparison of the OmpL2 sequence with tnat of known outer membraneproteins revealed areas of homology to the TonB-dependent outer membraneproteins. The TonB-dependent proteins form ligand-specific channels inthe outer membrane of gram-negative bacteria. Seven stretches ofsequence have been found to be conserved in all Ton B-dependent outermembrane proteins (Kadner, R. J., Molecular Microbiology, 4:2027-2033,1990). Sequence comparison, using the GAP program (Devereux, J., et al.,Nucl. Acids Res., 12:387-395, 1984) demonstrated that. the OmpL2sequence is homologous in all seven of the conserved regions.

The bacterial genes for the OmpL2 outer membrane protein can likely bederived from any strain of pathogenic Leptospira. Preferably the proteinis from Leptospira alstoni, strain RM52 (National LeptospirosisReference Laboratory, Ames, Iowa). Leptospira alstoni is the mostcurrent name for the pathogenic Leptospira previously grouped togetherin the family of Leptospira interrogans. The Leptospira interrogans arepublically available through the ATCC (Rockville, Md.), for example.

The invention provides polynucleotides encoding the Leptospira OmpL2protein. These polynucleotides include DNA and RNA sequences whichencode the protein. It is understood that all polynucleotides encodingall or a portion of OmpL2 are also included herein, so long as thesepolynucleotides exhibit the function of native or full length OmpL2,such as the ability to induce or bind antibody. Such polynucleotidesinclude both naturally occurring and intentionally manipulated, forexample, mutagenized polynucleotides.

DNA sequences of the invention can be obtained by several methods. Forexample, the DNA can be isolated using hybridization procedures whichare well known in the art. These include, but are not limited to: 1)hybridization of probes to genomic libraries to detect shared nucleotidesequences and 2) antibody screening of expression libraries to detectshared structural features.

Hybridization procedures are useful for the screening of recombinantclones by using labeled mixed synthetic oligonucleotide probes whereeach probe is potentially the complete complement of a specific DNAsequence in the hybridization sample which includes a heterogeneousmixture of denatured double-stranded DNA. For such screening,hybridization is preferably performed on either single-stranded DNA ordenatured double-stranded DNA. By using stringent hybridizationconditions directed to avoid non-specific binding, it is possible, forexample, to allow the autoradiographic visualization of a specific DNAclone by the hybridization of the target DNA to that single probe in themixture which is its complete complement (Wallace, et al., Nucleic AcidResearch, 9:879, 1981).

Alternatively, an expression library can be screened indirectly forOmpL2 peptides having at least one epitope using antibodies to OmpL2.Such antibodies can be either polyclonally or monoclonally derived andused to detect expression product indicative of the presence of OmpL2DNA. Generally, a lambda gt11 library is constructed and screenedimmunologically according to the method of Huynh, et al., (in DNACloning:A Practical Approach, D. M. Glover, ed., 1:49, 1985).

The development of specific DNA sequences encoding OmpL2 can also beobtained by: (1) isolation of a double-stranded DNA sequence from thegenomic DNA, and (2) chemical manufacture of a DNA sequence to providethe necessary codons for the polypeptide of interest.

DNA sequences encoding OmpL2 can be expressed in vitro by DNA transferinto a suitable host cell. “Recombinant host cells” or “host cells” arecells in which a vector can be propagated and its DNA expressed. Theterm also includes any progeny of the subject host cell. It isunderstood that not all prdgeny are identical to the parental cell sincethere may be mutations that occur at replication. However, such progenyare included when the terms above are used.

The term “host cell” as used in the present invention is meant toinclude not only prokaryotes, but also, such eukaryotes as yeasts,filamentous fungi, as well as plant and animal cells. The term“prokaryote” is meant to include all bacteria which can be transformedwith the gene for the expression of the OmpL2 outer membrane protein ofLeptospira. Prokaryotic hosts may include Gram negative as well as Grampositive bacteria, such as E. coli S. typhimurium, and Bacillussubtilis.

A recombinant DNA molecule coding for the OmpL2 protein can be used totransform a host using any of the techniques commonly known to those ofordinary skill in the art. Especially preferred is the use of a plasmidcontaining the OmpL2 coding sequence for purposes of prokaryotictransformation. Where the host is prokaryotic, such as E. coli,competent cells which are capable of DNA uptake can be prepared fromcells harvested after exponential growth phase and subsequently treatedby the CaCl₂ method by procedures well known in the art. Alternatively,MgCl₂ or RbCl can be used. Transformation can also be performed afterforming a protoplast of the host cell.

In the present invention, the OmpL2 sequences may be inserted into arecombinant expression vector. The term “recombinant expression vector”refers to a plasmid, virus or other vehicle known in the art that hasbeen manipulated by insertion or incorporation of OmpL2 geneticsequences. Such expression vectors contain a promoter sequence whichfacilitates the efficient transcription of the inserted genetic sequencein the host. The expression vector typically contains an origin ofreplication, a promoter, as well as specific genes which allowphenotypic selection of the transformed cells. The transformedprokarlotic hosts can be cultured according to means known in the art toachieve optimal cell growth. Various shuttle vectors for the expressionof foreign genes in yeast have been reported (Heinemann, et al., Nature,340:205, 1989; Rose, et al., Gene, 60:237, 1987). Biologicallyfunctional DNA vectors capable of expression and replication in a hostare known in the art. Such vectors are used to incorporate DNA sequencesof the invention.

Methods for preparing fused, operably linked genes and expressing themin bacteria are known and are shown, for example, in U.S. Pat. No.4,366,246 which is incorporated herein by reference. The geneticconstructs and methods described therein can be utilized for expressionof Leptospira OmpL2 in prokaryotic hosts.

Examples of promoters which can be used in the invention are: rec A,trp, lac, tac, and bacteriophage lambda p_(R) or p_(L). Examples ofplasmids which can be used in the invention are listed in Maniatis, etal., (Molecular Cloning, Cold Spring Harbor Laboratories, 1982).

Antibodies provided in the present invention are immunoreactive withOmpL2 protein. Antibody which consists essentially of pooled monoclonalantibodies with different epitopic specificities, as well as distinctmonoclonal antibody preparations are provided. Monoclonal antibodies aremade from antigen containing fragments of the protein by methods wellknown in the art (Kohler, et al., Nature, 256:495, 1975; CurrentProtocols in Molecular Biology, Ausubel, et al., ed., 1989).

The term “antibody” as used in this invention includes intact moleculesas wetl as fragments thereof, such as Fab, F(ab′)₂, and Fv which arecapable of binding the epitopic determinant. These antibody fragmentsretain some ability to selectively bind with its antigen of receptor andare defined as follows:

(1) Fab, the fragment which contains a monovalent antigen-bindingfragment of an antibody molecule can be produced by digestion of wholeantibody with the enzyme papain to yield an intact light chain and aportion of one heavy chain;

(2) Fab′, the fragment of an antibody molecule can be obtained bytreating whole antibody with pepsin, followed by reduction, to yield anintact light chain and a portion of the heavy chain; two Fab′ fragmentsare obtained per antibody molecule;

(3) (Fab′)₂, the fragment of the antibody that can be obtained bytreating whole antibody with the enzyme pepsin without subsequentreduction; F(ab′)₂ is a dimer of two Fab′ fragments held together by twodisulfide bonds;

(4) Fv, defined as a genetically engineered fragment containing thevariable region of the light chain and the variable region of the heavychain expressed as two chains; and

(5) Single chain antibody (“SCA”), defined as a genetically engineeredmolecule containing the variable region of the light chain, the variableregion of the heavy chain, linked by a suitable polypeptide linker as agenetically fused single chain molecule.

Methods of making these fragments are known in the art. (See forexample, Harlow and Lane, Antibodies: A Laboratory Manual, Cold SpringHarbor Laboratory, New York (1988), incorporated herein by reference).

As used in this invention, the term “epitope” means any antigenicdeterminant on an antigen to which the paratope of an antibody binds.Epitopic determinants usually consist of chemically active surfacegroupings of molecules such as amino acids or sugar side chains andusually have specific three dimensional structural characteristics, aswell as specific charge characteristics.

Antibodies which bind to the OmpL2 polypeptide of the invention can beprepared using an intact polypeptide or fragments containing smallpeptides of interest as the immunizing antigen. The polypeptide or apeptide of SEQ ID NO:2 used to immunize an animal can be derived fromtranslated cDNA or chemical synthesis which can be conjugated to acarrier protein, if desired. Such commonly used carriers which arechemically coupled to the peptide include keyhole limpet hemocyanin(KLH), thyroglobulin, bovine serum albumin (BSA), and tetanus toxoid.The coupled peptide is then used to immunize the animal (e.g., a mouse,a rat, or a rabbit).

If desired, polyclonal or monoclonal antibodies can be further purified,for example, by binding to and elution from a matrix to which thepolypeptide or a peptide to which the antibodies were raised is bound.Those of skill in the art will know of various techniques common in theimmunology arts for purification and/or concentration of polyclonalantibodies, as well as monoclonal antibodies (See for example, Coligan,et al., Unit 9, Current Protocols in Immunology, Wiley Interscience,1991, incorporated by reference).

It is also possible to use the anti-idiotype technology to producemonoclonal antibodies which mimic an epitope. For example, ananti-idiotypic monoclonal antibody made to a first monoclonal antibodywill have a binding domain in the hypervariable region which is the“image” of the epitope bound by the first monoclonal antibody.

Minor modifications of OmpL2 primary amino acid sequence may result inproteins which have substantially equivalent function compared to theOmpL2 protein described herein. Such modifications may be deliberate, asby site-directed mutgaenesis, or may be spontaneous. All proteinsproduced by these modifications are included herein as long as OmpL2function exists.

Modifications of OmpL2 primary amino acid sequence also includeconservative variations. The term “conservative variation” as usedherein denotes the replacement of an amino acid residue by another,biologically similar residue. Examples of conservative variationsinclude the substitution of one hydrophobic residue such as isoleucine,valine, leucine or methionine for another, or the substitution of onepolar residue for another, such as the substitution of arginine forlysine, glutamic for aspartic acids, or glutamine for asparagine, andthe like. The term “conservative variation” also includes the use of asubstituted amino acid in place of an unsubstituted parent amino acidprovided that antibodies raised to the substituted polypeptide alsoimmunoreact with the unsubstituted polypeptide.

Isolation and purification of microbially expressed protein, onfragments thereof, provided by the invention, may be carried out byconventional means including preparative chromatography andimmunological separations involving monoclonal or polyclonal antibodies.

The invention extends to any host modified according to the methodsdescribed, or modified by any other methods, commonly known to those ofordinary skill in the art, such as, for example, by transfer of geneticmaterial using a lysogenic phage, and which result in a prokaryoteexpressing the Leptospira gene for OmpL2 protein. Prokaryotestransformed with the Leptospira gene encoding the OmpL2 protein areparticularly useful for the production of polypeptides which can be usedfor the immunization of an animal (e.g., a rabbit).

In one embodiment, the invention provides a pharmaceutical compositionuseful for inducing an immune response to pathogenic Leptospira in ananimal comprising an immunologically effective amount of OmpL2 in apharmaceutically acceptable carrier. The term “immunogenically effectiveamount,” as used in describing the invention, is meant to denote thatamount of Leptospira antigen which is necessary to induce in an animalthe production of an immune response to Leptospira. The OmpL2 outermembrane protein of the invention is particularly useful in sensitizingthe immune system of an animal such that, as one result, an immuneresponse is produced which ameliorates the effect of Leptospirainfection.

The OmpL2 outer membrane protein can be administered parenterally byinjection, rapid infusion, nasopharyngeal absorption, dermal absorption,and orally. Pharmaceutically acceptable carrier preparations forparenteral administration include sterile or aqueous or non-aqueoussolutions, suspensions, and emulsions. Examples of non-aqueous solventsare propylene glycol, polyethylene glycol, vegetable oils such as oliveoil, and injectable organic esters such as ethyl oleate. Carriers forocclusive dressings can be used to increase skin permeability andenhance antigen absorption. Liquid dosage forms for oral administrationmay generally comprise a liposome solution containing the liquid dosageform. Suitable forms for suspending the liposomes include emulsions,suspensions, solutions, syrups, and elixirs containing inert diluentscommonly used in the art, such as purified water. Besides the inertdiluents, such compositions can also include adjuvants, wetting agents,emulsifying and suspending agents, and sweetening, flavoring, andperfuming agents.

It is also possible for the antigenic preparations containing the OmpL2protein of the invention to include an adjuvant. Adjuvants aresubstances that can be used to nonspecirfically augment a specificimmune response. Normally, the adjuvant and the antigen are mixed priorto presentation to the immune system, or presented separately, but intothe same site of the animal being immunized. Adjuvants can be looselydivided into several groups based on their composition. These groupsinclude oil adjuvants (for example, Freund's Complete and Incomplete),mineral salts (for example, AlK(SO₄)₂, AlNa(SO₄)₂, AlNH₄(SO₄), silica,alum, Al(OH)₃, Ca₃(PO₄)₂, kaolin, and carbon), polynucleotides (forexample, poly IC and poly AU acids), and certain natural substances (forexample, wax D from Mycobacterium tuberculosis, as well as substancesfound in Corynebacterium parvum, Bordetella pertussis, and members ofthe genus Brucella).

In another embodiment, a method of inducing an immune response topathogenic Leptospira in animal is provided. Many different techniquesexist for the timing of the immunizations when a multiple immunizationregimen is utilized. It is possible to use the antigenic preparation ofthe invention more than once to increase the levels and diversity ofexpression of the immune response of the immunized animal. Typically, ifmultiple immunizations are given, they will be spaced two to four weeksapart. Subjects in which an immune response to Leptospira is desirableinclude swine, cattle and humans.

Generally, the dosage of OmpL2 protein administered to an animal willvary depending on such factors as age, condition, sex and extent ofdisease, if any, and other variables which can be adjusted by one ofordinary skill in the art.

The antigenic preparations of the invention can be administered aseither single or multiple dosages and can vary from about 10 ug to about1,000 ug for the Leptospira OmpL2 antigen per dose, more preferably fromabout 50 ug to about 700 ug OmpL2 antigen per dose, most preferably fromabout 50 ug to about 300 ug OmpL2 antigen per dose.

When used for immunotherapy, the monoclonal antibodies of the inventionmay be unlabeled or labeled with a therapeutic agent. These agents canbe coupled either directly or indirectly to the monoclonal antibodies ofthe invention. One example of indirect coupling is by use of a spacermoiety. These spacer moieties, in turn, can be either insoluble orsoluble (Diener, et al., Science, 231:148, 1986) and can be selected toenable drug release from the monoclonal antibody molecule at the targetsite. Examples of therapeutic agents which can be coupled to themonoclonal antibodies of the invention for immunotherapy are drugs,radioisotopes, lectins, and toxins.

The labeled or unlabeled monoclonal antibodies of the invention can alsobe used in combination with therapeutic agents such as those describedabove. Especially preferred are therapeutic combinations comprising themonoclonal antibody of the invention and immunomodulators and otherbiological response modifiers.

When the monoclonal antibody of the invention is used in combinationwith various therapeutic agents, such as those described herein, theadministration of the monoclonal antibody and tile therapeutic agentusually occurs substantially contemporaneously. The term “substantiallycontemporaneously” means that the monoclonal antibody and thetherapeutic agent are administered reasonably close together withrespect to time. Usually, it is preferred to administer the therapeuticagent before the monoclonal antibody. For example, the therapeutic agentcan be administered 1 to 6 days before the monoclonal antibody. Theadministration of the therapeutic agent can be daily, or at any otherinterval, depending upon such factors, for example, as the nature of thedisorder, the condition of the patient and half-life of the agent.

The dosage ranges for the administration of monoclonal antibodies of theinvention are those large enough to produce the desired effect in whichthe onset symptoms of the leptosporal disease are ameliorated. Thedosage should not be so large as to cause adverse side effects, such asunwanted cross-reactions, anaphylactic reactions, and the like.Generally, the dosage will vary with the age, condition, sex and extentof the disease in the subject and can be determined by one of skill inthe art. The dosage can be adjusted by the individual physician in theevent of any complication. Dosage can vary from about 0.1 mg/kg to about2000 mg/kg, preferably about 0.1 mg/kg to about 500 mg/kg, in one ormore dose administrations daily, for one or several days. Generally,when the monoclonal antibodies of the invention are administeredconjugated with therapeutic agents, lower dosages, comparable to thoseused for in vivo diagnostic imaging, can be used.

The monoclonal antibodies of the invention can be administeredparenterally by injection or by gradual perfusion over time. Themonoclonal antibodies of the invention can be administeredintravenously, intraperitoneally, intramuscularny, subcutaneously,intracavity, or transdermally, alone or in combination with effectorcells.

Preparations for parenteral administration include sterile aqueous ornon-aqueous solutions, suspensions, and emulsions. Examples ofnon-aqueous solvents are propylene glycol, polyethylene glycol,vegetable oils such as olive oil, and injectable organic esters such asethyl oleate. Aqueous carriers include water, alcoholic/aqueoussolutions, emulsions or suspensions, including saline and bufferedmedia. Parenteral vehicles include sodium chloride solution, Ringer'sdextrose, dextrose and sodium chloride, lactated Ringer's intravenousvehicles include fluid and nutrient replenishers, electrolytereplenishers (such as those based on Ringer's dextrose), and the like.Preservatives and other additives may also be present such as, forexample, antimicrobials, anti-oxidants, chelating agents and inert gasesand the like.

In a further embodiment, the invention provides a method of detecting apathogenic Leptospira-associated disorder in a subject comprisingcontacting a cell component with a reagent which binds to the cellcomponent. The cell component can be nucleic acid, such as DNA or RNA,or it can be protein. When the component is nucleic acid, the reagent isa nucleic acid probe or PCR primer. When the cell component is protein,the reagent is an antibody probe. The probes are detectably labeled, forexample, with a radioisotope, a fluorescent compound, a bioluminescentcompound, a chemiluminescent compound, a metal chelator or an enzyme.Those of ordinary skill in the art will know of other suitable labelsfor binding to the antibody, or will be able to ascertain such, usingroutine experimentation.

For purposes of the invention, an antibody or nucleic acid probespecific for OmpL2 may be used to detect the presence of OmpL2polypeptide (using antibody) or polynucleotide (using nucleic acidprobe) in biological fluids or tissues. Any specimen containing adetectable amount of OmpL2 antigen or polynucleotide can be used. Apreferred specimen of this invention is blood, urine, cerebrospinalfluid, or tissue of endothelial origin.

When the cell component is nucleic acid, it may be necessary to amplifythe nucleic acid prior to binding with a Leptospira specific probe.Preferably, polymerase chain reaction (PCR) is used, however, othernucleic acid amplification procedures such as ligase chain reaction(LCR), ligated activated transcription (LAT) and nucleic acidsequence-based amplification (NASBA) may be used.

Another technique which may also result in greater sensitivity consistsof coupling antibodies to low molecular weight haptens. These haptenscan then be specifically detected by means of a second reaction. Forexample, it is common to use such haptens as biotin, which reacts withavidin, or dinitrophenyl, pyridoxal, and fluorescein, which can reactwith specific antihapten antibodies.

Alternatively, OmpL2 polypeptide can be used to detect antibodies toOmpL2 polypeptide in a specimen. The OmpL2 of the invention isparticularly suited for use in immunoassays in which it can be utilizedin liquid phase or bound to a solid phase carrier. In addition, OmpL2used in these assays can be detectably labeled in various ways.

Examples of immunoassays which can utilize the OmpL2 of the inventionare competitive and noncompetitive immunoassays in either a direct orindirect format. Examples of such immunoassays are the radioimmunoassay(RIA), the sandwich (immunometric assay) and the Western blot assay.Detection of antibodies which bind to the OmpL2 of the invention can bedone utilizing immunoassays which run in either the forward, reverse, orsimultaneous modes, including immunohistochemical assays onphysiological samples. The concentration of OmpL2 which is used willvary depending on the type of immunoassay and nature of the detectablelabel which is used. However, regardless of the type of immunoassaywhich is used, the concentration of OmpL2 utilized can be readilydetermined by one of ordinary skill in the art using routineexperimentation.

The OmpL2 of the invention can be bound to many different carriers andused to detect the presence of antibody specifically reactive with thepolypeptide. Examples of well-known carriers include glass, polystyrene,polyvinyl chloride, polypropylene, polyethylene, polycarbonate, dextran,nylon, amyloses, natural and modified celluloses, polyacrylamides,agaroses, and magnetite. The nature of the carrier can be either solubleor insoluble for purposes of the invention. Those skilled in the ar willknow of other suitable carriers for binding OmpL2 or will be able toascertain such, using routine experimentation.

There are many different labels and methods of labeling known to thoseof ordinary skill in the art. Examples of the types of labels which canbe used in the present invention include enzymes, radioisotopes,colloidal metals, fluorescent compounds, chemiluminescent compounds, andbioluminescent compounds.

For purposes of the invention, the antibody which binds to OmpL2 of theinvention may be present in various biological fluids and tissues. Anysample containing a detectable amount of antibodies to OmpL2 can beused. Normally, a sample is a liquid such as urine, saliva,cerebrospinal fluid, blood, serum and the like, or a solid or semi-solidsuch as tissue, feces and the like.

The monoclonal antibodies of the invention, directed toward OmpL2, arealso useful for the in vivo detection of antigen. The detectably labeledmonoclonal antibody is given in a dose which is diagnosticallyeffective. The term “diagnostically effective” means that the amount ofdetectably labeled monoclonal antibody is administered in sufficientquantity to enable detection of Leptospira OmpL2 antigen for which themonoclonal antibodies are specific.

The concentration of detectably labeled monoclonal antibody which isadministered should be sufficient such that the binding to those cells,body fluid, or tissue having OmpL2 is detectable compared to thebackground. Further, it is desirable that the detectably labeledmonoclonal antibody be rapidly cleared from the circulatory system inorder to give the best target-to-background signal ratio.

As a rule, the dosage of detectably laieled monoclonal antibody for invivo diagnosis will vary depending on such; factors as age, sex, andextent of disease of the subject. The dosage of monoclonal antibody canvary from about 0.001 mg/m² to about 500 mg/m², preferably 0.1 mg/m² toabout 200 mg/m², most preferably about 0.1 mg/m² to about 10 mg/m². Suchdosages may vary, for example, depending on whether multiple injectionsare given, and other factors known to those of skill in the art.

For in vivo diagnostic imaging, the type of detection instrumentavailable is a major factor in selecting a given radioisotope. Theradioisotope chosen must have a type of decay which is detectable for agiven type of instrument. Still another important factor in selecting aradioisotope for in vivo diagnosis is that the half-life of theradioisotope be long enough so that it is still detectable at the timeof maximum uptake by the target, but short enough so that deleteriousradiation with respect to the host is minimized. Ideally, a radioisotopeused for in vivo imaging will lack a particle emission, but produce alarge number of photons in the 140-250 key range, which may be readilydetected by conventional gamma cameras.

For in vivo diagnosis, radioisotopes may be bound to immunoglobulineither directly or indirectly by using an intermediate functional group.Intermediate functional groups which often are used to bindradioisotopes which exist as metallic ions to immunoglobulins are thebifunctional chelating agents such as diethylenetriaminepentacetic acid(DTPA) and ethylenediaminetetraacetic acid (EDTA) and similar molecules.Typical examples of metallic ions which can be bound to the monoclonalantibodies of the invention are ¹¹¹In, ⁹⁷Ru, ⁶⁷Ga, ⁶⁸Ga, ⁷²As, ⁸⁹Zr, and²⁰¹Tl.

The moroclonal anticodies of the invention can also so be labeled with aparamagnetic isotope for purposes of in vivo diagnosis, as in magneticresonance imaging (MRI) or electron spin resornano (ES). In general, anyconventional method for visualizing diagnostic imaging can be utilized.Usually gamma and positron emitting radioisotopes are used for cameraimaging and paramagnetic isotopes for MRI. Elements which areparticularly useful in such techniques include ¹⁵⁷Gd, ⁵⁵Mn, ¹⁶²Dy, ⁵²Cr,and ⁵⁶Fe.

The monoclonal antibodies of the invention can be used to monitor thecourse of amelioration of Leptospira associated disorder. Thus, bymeasuring the increase or decrease of Leptospira OmpL2 polypeptide orantibodies to OmpL2 polypeptide present in various body fluids ortissues, it would be possible to determine whether a particulartherapeutic regiment aimed at ameliorating the disorder is effective.

The materials for use in the method of the invention are ideally suitedfor the preparation of a kit. Such a kit may comprise a carrier meansbeing compartmentalized to receive in close confinement one or morecontainer means such as vials, tubes, and the like, each of thecontainer means comprising one of the separate elements to be used inthe method. For example, one of the container means may comprise a OmpL2binding reagent, such as an antibody. A second container may furthercomprise OmpL2 polypeptide. The constituents may be present in liquid orlyophilized form, as desired.

The following examples are intended to illustrate but not limit theinvention. While they are typical of those that might be used, otherprocedures known to those skilled in the art may alternatively be used.

EXAMPLES

The following examples describe the identification of OmpL2 as animportant leptospiral outer membrane protein. The method by which theompL2 gene was cloned and sequenced is described. Sequence analysis andhomology studies are shown, further indicating that OmpL2 is an outermembrane protein of pathogenic Leptospira and therefore is an excellentvaccine candidate.

Example 1

CLONING OF ompL2

The ompL2 gene was identified using an approach for identification ofgenes encoding exported leotospiral proteins by screening for blue-halocolonies using the pMG expresssion vector and E. coli KS330 (Blanco, etal., Molecular Microbiology, 5:2405, 1991; Giladi, et al., J. Bacteriol,75:4129,1993). The pMG vector is a phoA expression vector, which, likeTnphoA, is useful in identifying genes encoding membrane-spanningsequences or signal peptides. This cloning system has been modified tofacilitate the distinction of outer membrane and periplasmic alkalinephosphatase (AP) fusion proteins from inner membrane AP fusion proteinsby transforming pMG recombinants into E. coli KS330, the strain firstused in the “blue halo” assay described by Strauch and Beckwith (Proc.Natl. Acad. Sci., USA 85:1576, 1988). The lipoprotein mutation Ipp-5508of KS330 results in an outer membrane that is leaky to macromolecules,and its degP4 mutation greatly reduces periplamic proteolyticdegradation of AP fusion proteins. pMG AP fusions containing cleavablesignal peptides, including the E. coli periplasmic protein β-lactamase,OmpA and MOMP and Tp9, a Treponema palladum AP recombinant, have beenshown to diffuse through the leaky outer membrane protein of KS330 andresult in blue colonies with blue halos (Giladi, et al., supra). Incontrast, inner membrane AP fusions derived from E.coli proteins,including leader peptidase, SecY, and the tetracycline resistance geneproduct, resulted in blue colonies without blue halos. The pMG/KS330r-cloning and screening approach identifies genes encoding proteins withcleavable signal peptides and therefore is useful in the identificationof genes encoding potential virulence factors.

Escherichia coli strains were grown at 37° C. on Luria-Bertani medium.All restriction endonucleases and DNA-modifying enzymes were used inaccordance with the specifications of the manufacturer (BethesdaResearch Laboratories, Inc., Gaithersburg, Md., or Boehringer MannheimBiochemicals, Indianapolis, Ind.).

L. aistoni strain RM52 (National Leptospirosis Reference Laboratory,Ames, Iowa) genomic DNA was prepared by the method of Yelton, D. B., andN. W. Charon, (Gene, 28:147, 1984). Genomic DNA was partially digestedwith Sau3A to a mean size of about 3.0 kb, ligated to BamHl-digested pMGand transformed into KS330r-. Approximately, 80,000 recombinant cloneswere screened on XP-IPTG-containing plates (Giladi, et al., supra), andabout 10,000 clones were screened on XP plates without IPTG, yielding226 blue colonies. Clones producing blue colonies were subcultured andspotted on high IPTG, high XP plates resulting in blue colonies, 66 ofwhich showed blue halo formation. One such clone showing a blue halo,designated L2.086, was chosen for further study. This clone contained a237 bp insert in pMG. The clone was identified as an outer membraneprotein since it contained a leader sequence and leader peptidase Icleavage site (as determined from nucleic and deduced amino acidsequence) as indicated in FIG. 1 (↑).

The remainder of the ompL2 gene was cloned on 3.0 kb EcoRI fragment. Alibrary of the DNA from L. alstoni was generated in the λ Zap II vectorsystem (Stratagene, San Diego, Calif.). Following digestion with EcoRI,the DNA fragments were ligated into the phage vector. The library waspackaged and plated according to the manufacturer's recommendations.Approximately 10,000 plaques were plated, transferred to filters induplicate, and processed as previously described (Maniatis, et al.,Molecular Cloning: A Laboratory Manual, Cold Spring Harbor, N.Y., 1982).An oligonucleotide probe based on the L2.086 insert was radiolabled asdescribed (Maniatis, et al., supra) and used for plaque hybridizations.Positive recombinant pBluescript SK(−) clones were recovered by in vivoexcision according to the manufacturer's instructions.

Example 2

SEQUENCE ANALYSIS FOR OmpL2

The L2.086 insert was sequenced in pMG by using the dideoxynucleotidechain termination method described by Sanger, et al., (Proc. Natl. Acad.Sci. USA, 74:5463, 1977) and [α-³⁵S]-dATP (See Giladi, et al., supra).The remainder of the ompL2 gene was sequenced using standard M13 primersand custom oligonucleotide primers synthesized at UCLA, Dept. ofMicrobiology & Immunology for sequencing double-stranded templates.Sequencing reactions were performed for both strands using the Deaza T7Sequencing kit protocol as described by Pharmacia Biotech, Inc., and[α-³⁵S]dATP (specific activity, 1,000 Ci/mmol). DNA and deduced aminoacid sequences were analyzed using DNA Strider 1.0 (Marck, C., Nucl.Acids Res. 16:1829, 1988). Protein homology searches were performed withthe Profilesearch and FASTA programs found in the University ofWisconsin Genetics Computer Group (GCG), Inc., package, ver. 7.0(Devereux, et al., Nucl. Acids Res. 12:387, 1984).

An open reading frame of 1740 bp was identified, which would encode a540-amino-acid polypeptide with a predicted molecular mass of 63-kDa(FIG. 1). A Shine-Dalgarno ribosome binding site (RBS) was identifiedupstream from the ATG start codon, as well as putative −35 and −10promoter regions. The TAA stop codon is indicated by an asterisk. Database searching using the FASTA and ProfileSearch programs failed toreveal significant amino acid homologies. However, secondary structureanalysis predicted numerous areas of amphipathic beta-sheets, consistentwith outer membrane protein transmembrane segments. Of special note isthe carboxy-terminal phenylalanine, a feature which is highly conservedamong outer membrane proteins (Struyve, M., et al., J. Mol. Biol.,218:141-148, 1991).

Comparison of the OmpL2 sequence with that of known outer membraneproteins revealed areas of homology to the TonB-dependent outer membraneproteins. The TonB-dependent proteins form ligand-specific channels inthe outer membrane of gram-negative bacteria. Seven stretches ofsequence have been found to be conserved in all Ton B-dependent outermembrane proteins (Kadner, R. J., Molecular Microbiology, 4:2027-2033,1990). Sequence comparison, using the GAP program (Devereux, J., et al.,Nucl. Acids Res., 12:387-395, 1984) demonstrated that the OmpL2 sequenceis homologous in all seven of the conserved regions (FIG. 2). Peptidealignment between OmpL2 and eight TonB-dependent outer membraneproteins, for all seven regions of homology identified by Kadner, supra.Domain 1 is the “TonB box” which has been implicated in the directinteraction of Ton B with outer membrane receptors. OmpL2 is alignedwith TBP1 (N. gonorrhoeae transferrin-binding protein 1); BtuB (E. colivitamin B₁₂ receptor); Cir (E. coli colicin I receptor); Iuta (E. coliaerobactin receptor); FhuA (E. coli ferrichrome receptor); PupA (P.putida pseudobactin receptor); IrgA (V. cholerae iron-regulated outermembrane protein); FoxA (Y. enterocolitica ferrioxamine receptor).Asterisks mark positions of complete identity in all nine proteins.Positions are indicated where OmpL2 has a functionally similar aminoacid as all (|), half (:), or 25% (.) of the other eight proteins, aspredicted by the Mutation Matrix of Dayhoff. (In M. O. Dayhoff (ed.),Atlas of protein sequence and Structure, Vol. 5, Suppl. 3, NationalBiomedical Research Fdn., Washington, D.C.).

The first of these segments is known as the TonB box, which ischaracterized by the following consensus sequence: Thr-X-Y-Val. TheOmpL2 TonB box retains the Threonine, but there is a conservativesubstitution of Isoleucine for Valine. A substitution at this positionis unprecidented among the known TonB-dependent outer membrane proteins,however, spirochetes occupy one of the deepest branches in eubacterialevolution and OmpL would be the first spirochetal TonB-dependent outermembrane protein to be identified. Mutagenesis studies demonstrate thatinteraction of TonB-dependent outer membrane proteins with TonB arehighly tolerant of amino acid substitutions within the TonB box, evenaat the invariant Valine positions (Gudmundsdottir, A., et al., Jourmalof Bacteriology, 171:6526-6533, 1989).

Example 3

TOPOLOGY OF OmpL2

The topology of the E. coli TonB-dependent outer membrane protein, FepA,has been studied using monoclonal antibodies and deletion mutagenesis(Rutz, J. M., et al., Science, 258:471-474, 1992). A topology for the Y.enterocolica TonB-dependent outer membrane protein, FoxA, has also beenproposed (Baumler, A. J., et al., Molecular Microbiology, 6:1309-1321,1992). The OmpL2 sequence contains 24 stretches of amphipathicbeta-sheets, consistent with transmembrane segments, making it possibleto propose a topological model with large surface-exposed loops andshort periplasmic loops typical of outer membrane proteins (FIG. 3). Themembrane-spanning beta-sheets are shown within rectangles in a staggeredarray with the hydrophobic, membrane-facing residues on the right sideof the array.

Example 4

EXPRESSION OF ompL2 DURING IRON DEPLETION

Studies show that OmpL2 is produced in greater amounts by L. alstoniwhen grown in iron-depleted media (bovuminar (Invirogen, N.Y.)containing 50 μM dipyridyl, an iron chelator). There is a potentialFur-binding site in the promoter region upstream of the ompL2 gene,which would also indicate that expression of ompL2 is turned on iniron-limiting conditions. This suggests that expression of OmpL2 occurswhen Leptospira are in the host, a feature common to most of the Ton-Bdependent outer membrane proteins. An outer membrane protein which isproduced by a bacterial pathogen when it enters the host would be anideal vaccine candidate.

Example 5

SOUTHERN and NORTHERN BLOT ANALYSIS

Southern blot analysis is performed as described previously by Maniatis,et al., supra. A probe from ompL2 is labeled at its 5′ end with[γ-³²P]ATP (5,000 Ci/mmol; Amersham Corp., Arlington Heights, Ill.) andT4 polynucleotide kinase followed by purification over a BioSpin 6column (Bio-rad Laboratories, Hercules, Calif.). Membranes containingDNA from various Leptospira species are hybridized overnight at 37° C.with 1×10⁶ cpm/ml of hybridization buffer.

For Northern blot analysis, total cellular RNA is isolated from L.aistoni by the method as previously described (Maniatis, et al., supra).Approximately 15 μg of RNA is electrophoresed in duplicate throuch a1.5% agarose-formaldehyde gel and transferred to nitrocellulose. Thefilters are probed with PCR-generated DNA fragments of ompL2 generadiolabled with [α-³²P]dATP using the Random Primers DNA LabelingSystem (BRL). Hybridizations are conducted as previously described(Maniatis, et al., supra).

Example 6

CLONING OF THE ompL2 GENE INTO THE pRCET EXPRESSION VECTOR

The pBluescript plasmid containing the ompL2 gene was digested withHincII and ClaI. The resulting DNA fragment encoding thecarboxy-terminal half of the OmpL2 protein was isolated by agarose gelelectrophoresis, and ligated into pRSET (Invitrogen, San Diego, Calif.)digested with PvuIII and Csp45I. The resulting construct, pRSET-ompL2,encodes a fusion protein containing a 41 amino acid His6 binding site atthe amino terminus of OmpL2. The six histidines allow for pH-dependentaffinity purification of the fusion protein on a nickel resin column tothe exclusion of E. coli proteins. The pRSET fusion protein is under T7promoter control. After transformation of pRSET-ompL2 into E. coli DH5α,milligram quantities of the His6-OmpL2 fusion protein are produced inthe presence of isopropyl-β-D-thiogalactoside (IPTG, Sigma).

Example 7

IMMUNIZATION OF RABBITS WITH PURIFIED OmpL2

The His6-OmpL2 fusion protein is separated from other insolublematerials by SDS-PAGE. The His6-OmpL2 band containing about 50micrograms of protein is cut out of the acrylamide gel, dessicated,ground to powder, mixed with Freund's complete adjuvant and inoculatedsubcutaneously and intramuscularly into a New Zealand White male rabbit.Additional His6-OmpL2 fusion protein is solubilized in 6M guanidine andpurified over the nickel resin column and dialyzed in 10 mM Tris, pH8.0. The secondary immunization is given six weeks after the primaryimmunization using roughly 50 micrograms of purified His6-OmpL2 fusionprotein in Freund's incomplete adjuvant. The rabbit is bled two weeksafter the secondary immunization. The post-boost antiserum will reactwith the 63-kDa antigen on immunoblots of whole L. alstoni separated bySDS-PAGE. Immunoblots of L. alstoni fractioned with TX-114 revealreactivity with the 63-kDa OmpL2 antigen in the whole organism anddetergent phase, but not the aqueous phase or insoluble pellet.

Example 8

SURFACE LOCALIZATION WITH IMMUNOELECTRON MICROSCOPY

Having obtained a highly specific immunological reagent for localizationstudies, preliminary immunoelectron microscopy experiments can beconducted. A 20 μl suspension of 10⁷ L. alstoni is added to 0.5 ml ofheat-inactivated anti-OmpL2 antiserum or preimmune serum from the samerabbit and incubated for one hour with mixing. The bacteria are fixedfor 30 minutes by addition of 250 μl of 0.75% glutaraldehyde in 100 mMcacodylate buffer, pH 7.0. The bacteria are washed, applied to electronmicroscopy grids, and probed with protein G-colloidal gold (10 nmparticles).

Example 9

EXPRESSION OF OmpL2 WITH THE pTrc 99A EXPRESSION VECTOR

The His6 fusion protein is well suited for purification, but is notappropriate for immunoblotting studies because of the potential forbackground reactivity to the 41 additional amino acids containing theHis6 binding site. Preimmune sera from one of the rabbits reacts withthe His6-OmpL2 fusion protein, but not with native OmpL2. A BgI II-HindII fragment is isolated from the pRCET-ompL2 vector by gelelectrophoresis and cloned into the pTrc99A expression vector(Pharmacia) which had been reading frame adjusted with a 10-mer Nco Ilinker. The pTtrc99A-ompL2 construct, transformed into E. coli DH5αexpresses the entire mature OmpL2 protein, plus a start methionine andonly five additional amino acids supplied by the vector. E. coli DH5αcontaining the original pTrc99A vector serves as a negative control.Bacterial proteins are separated by SDS-PAGE and transferred tonitrocellulose, and probed with antisera from rabbits immunized with avariety of pathogenic Leptospira strains (antisera supplied by Dr.Arnold Kaufmann, Centers for Disease Control, Atlanta). Reactivity toOmpL2 is likely demonstrated with antisera to L. interrogans, serovarsicterohaemorrhagiae, pomona, and bratislava, L. alstoni, serovarsgrippotyphosa and Mozdok, L. santarosai, serovars bakeri and canalzonae,and L. weilii, serovar celledoni. OmpL2 is likely not only expressed,but also antigenically conserved among pathogenic Leptospira, a featurethat would make it an excellent vaccine candidate.

The foregoing is meant to illustrate, but not to limit, the scope of theinvention. Indeed, those of ordinary skill in the art can readilyenvision and produce further embodiments, based on the teachings herein,without undue experimentation.

SUMMARY OF SEQUENCES

SEQ ID NO:1 is the nucleotide sequence and deduced amino acid sequenceof ompL2.

SEQ ID NQ:2 is the deduced amino acid sequence of OmpL2.

10 1991 base pairs nucleic acid single linear DNA (genomic) not providedOmpL2 CDS 96..1715 1 GATCTTCATT TCTTTCCGAA AATTAAGTAA GACTTTATTTGTAAGGAGAG TGTAGCGGGA 60 TTTTCTAAGG AATTTTCGGT TTAAATCAAT CTGAC ATG ACCAAA CGT TCT AAA 113 Met Thr Lys Arg Ser Lys 1 5 TAC CTT TTC CTA TTT TTATTT CTT TTC TTT GGA ATC CAA ACT GGA ATT 161 Tyr Leu Phe Leu Phe Leu PheLeu Phe Phe Gly Ile Gln Thr Gly Ile 10 15 20 CAA GCA CAA CTT TGG ATT CCACCG GGT AGA CAG TAT ATG CAT CCC ACA 209 Gln Ala Gln Leu Trp Ile Pro ProGly Arg Gln Tyr Met His Pro Thr 25 30 35 GAG CCG TTT ACT TAT GAC CTT GGGATC AAT AAA TAT CAG AAA GAT TAT 257 Glu Pro Phe Thr Tyr Asp Leu Gly IleAsn Lys Tyr Gln Lys Asp Tyr 40 45 50 TAT CTC TAT GTG GCG CCT ACC GTC AATTTG AAC TTC GGA GGC GAT TTC 305 Tyr Leu Tyr Val Ala Pro Thr Val Asn LeuAsn Phe Gly Gly Asp Phe 55 60 65 70 GGA GCC TCT CTG ACT TTA CCT TTA AATTTT TTG ATC TAC GAT ACG GAG 353 Gly Ala Ser Leu Thr Leu Pro Leu Asn PheLeu Ile Tyr Asp Thr Glu 75 80 85 CCG AAA CAA GAA AAT TCT AGG ATC GGA AAGCTT AGG TCT TTC GAT TAC 401 Pro Lys Gln Glu Asn Ser Arg Ile Gly Lys LeuArg Ser Phe Asp Tyr 90 95 100 AAT GAC AAA AGC GAT TAT CTT AGA TTG ATCAAT AAT ATT TGG TTT GGC 449 Asn Asp Lys Ser Asp Tyr Leu Arg Leu Ile AsnAsn Ile Trp Phe Gly 105 110 115 CAG TAT GGA AAA TAC ACT CCC GGA GAA ATTACA TAT TCT GCA TCT TTA 497 Gln Tyr Gly Lys Tyr Thr Pro Gly Glu Ile ThrTyr Ser Ala Ser Leu 120 125 130 GGA AAA CTA TTC GAT GGT TAT ATA GGT CACGGA ACG ATC GTA AAC CGG 545 Gly Lys Leu Phe Asp Gly Tyr Ile Gly His GlyThr Ile Val Asn Arg 135 140 145 150 TAC GTA AAC AAT CAA CGT TTG GAT GTGTAT AAC GTA GGT CTT CAA GCA 593 Tyr Val Asn Asn Gln Arg Leu Asp Val TyrAsn Val Gly Leu Gln Ala 155 160 165 GAT ATA AAC AGT GAC TTT GGA GGA GTGCAG GTA TTT TCT AAT TCG ATC 641 Asp Ile Asn Ser Asp Phe Gly Gly Val GlnVal Phe Ser Asn Ser Ile 170 175 180 TAT ACG AGA GAA GTC AGT TCA GCA AGGGTT TAT ATC CGG CCC TTT GCC 689 Tyr Thr Arg Glu Val Ser Ser Ala Arg ValTyr Ile Arg Pro Phe Ala 185 190 195 GTT GGA TAT AAA CTT TTT GAT ATT GTTACC GGC CGG TCC AAA TTT TTG 737 Val Gly Tyr Lys Leu Phe Asp Ile Val ThrGly Arg Ser Lys Phe Leu 200 205 210 ACG ATG ATG ACA ATC GCA CAA GGA AACGTA GCA GAC GAG GCT GGA AGA 785 Thr Met Met Thr Ile Ala Gln Gly Asn ValAla Asp Glu Ala Gly Arg 215 220 225 230 AGA AAA GTT TAT GAA GAA GTA GGGGCG GAA GAA AAG GAA TCT TAT CGC 833 Arg Lys Val Tyr Glu Glu Val Gly AlaGlu Glu Lys Glu Ser Tyr Arg 235 240 245 GCT TTG ATC GAG GAT CAG AAG ACGCAC CAC AAA AAA GAA GAG ATG ATT 881 Ala Leu Ile Glu Asp Gln Lys Thr HisHis Lys Lys Glu Glu Met Ile 250 255 260 CCT GTG GAT AAG AAA CCG GAA AAACCT CGA AAT TTA AAA GAA ATA TTT 929 Pro Val Asp Lys Lys Pro Glu Lys ProArg Asn Leu Lys Glu Ile Phe 265 270 275 AAT CAA GAT AAT TGG GTT AAC CGGTTT GCA ATT GGT TAT ACG ACT GCG 977 Asn Gln Asp Asn Trp Val Asn Arg PheAla Ile Gly Tyr Thr Thr Ala 280 285 290 TTT GAT ACC AAA GCC CCT TCG GAACTT AAG TTT GAT ACG ACT GGA AAA 1025 Phe Asp Thr Lys Ala Pro Ser Glu LeuLys Phe Asp Thr Thr Gly Lys 295 300 305 310 TTG AGA GTG GAT GAA AAC GACAAT CCA CTC GTC AAG TCT ACG GAA AGA 1073 Leu Arg Val Asp Glu Asn Asp AsnPro Leu Val Lys Ser Thr Glu Arg 315 320 325 CTT TCG ATC ACT GGT TTC GATTTC GAA TAT AAA TTA CTC AGT GCG AAA 1121 Leu Ser Ile Thr Gly Phe Asp PheGlu Tyr Lys Leu Leu Ser Ala Lys 330 335 340 TAT ATA GAA CTG ACT CCC TATTAC GAC GTA AAT AAA ATC AAA CAG ATA 1169 Tyr Ile Glu Leu Thr Pro Tyr TyrAsp Val Asn Lys Ile Lys Gln Ile 345 350 355 GAA AAC GCA AAA GGT ACA CATTAC GGA GCG ATT CTT CGA TTG GGT GGA 1217 Glu Asn Ala Lys Gly Thr His TyrGly Ala Ile Leu Arg Leu Gly Gly 360 365 370 AAG GAC ATT TAT GTA CAA ATAAAA CCT GAA TAT AGA AAT ATG ACT GCA 1265 Lys Asp Ile Tyr Val Gln Ile LysPro Glu Tyr Arg Asn Met Thr Ala 375 380 385 390 ACG TAT ATT CCT ATG TATTTT GAT AGT TTT TAC GAA TTG GAA AGG TTT 1313 Thr Tyr Ile Pro Met Tyr PheAsp Ser Phe Tyr Glu Leu Glu Arg Phe 395 400 405 CAG AGT AAT TTA CAA AGTCAT ATT CCG CAG ACT AAA TTA GAA GCC CCA 1361 Gln Ser Asn Leu Gln Ser HisIle Pro Gln Thr Lys Leu Glu Ala Pro 410 415 420 AAA TTA GCC GAT CCG GATGGA TCT AAG ATA AAA GGA CAT TTT ACA CCT 1409 Lys Leu Ala Asp Pro Asp GlySer Lys Ile Lys Gly His Phe Thr Pro 425 430 435 GTA TTA TTC AAC TTT TATAGA TTT GCG ATT GAA TCG AAT TAC GAG AAT 1457 Val Leu Phe Asn Phe Tyr ArgPhe Ala Ile Glu Ser Asn Tyr Glu Asn 440 445 450 TAT TCC GGG CCG AAT AACTCT AGA GTA TTT TTA GGA GTT TAT ATT CCG 1505 Tyr Ser Gly Pro Asn Asn SerArg Val Phe Leu Gly Val Tyr Ile Pro 455 460 465 470 CTT GGA AGT ATG TTCCTA ATT AAT GGA TAT TAT ATG AAA AAA GCT TTT 1553 Leu Gly Ser Met Phe LeuIle Asn Gly Tyr Tyr Met Lys Lys Ala Phe 475 480 485 AAA TTA GAC GAT CGATCT CAA GGG GCC TTA GAA TTG GCG ATC AAT TTG 1601 Lys Leu Asp Asp Arg SerGln Gly Ala Leu Glu Leu Ala Ile Asn Leu 490 495 500 GGG CTT GTA ACA GTTAGG CTT CAG AAT ATA CGT AAA TGG GTT TAT GAT 1649 Gly Leu Val Thr Val ArgLeu Gln Asn Ile Arg Lys Trp Val Tyr Asp 505 510 515 ACG GCT TCT AGT CAATAC GAA GCC CAA GAC GAA CAG AAG ATA TTA TTT 1697 Thr Ala Ser Ser Gln TyrGlu Ala Gln Asp Glu Gln Lys Ile Leu Phe 520 525 530 TCC GGT GGT TTA TATTTT TAAAAAAGTA TTTTTTCTTC AAGTCTTGCG 1745 Ser Gly Gly Leu Tyr Phe 535540 AGTAAAAATG CAAAAGCTGT TTCTGTACGA AGAACTCGAT CGGAAAGATT TAATTTTTTG1805 AAACCGAAAC GTTTCCAAAA ATCGATTTCG TTTGGAACAA ATCCACTTTC CGGACCGATC1865 GCGGATAAAA TACGAGGTAT TTTAGAATAC ATTCCAAAAT TTGAATCTAA TTTTTTTTCT1925 TTAAACATCT GGGTAAAAGT AAAACCTTTT CGATCTAAAA CAAAACGAAA CGTAAAGTCT1985 AATTCT 1991 540 amino acids amino acid linear protein not provided2 Met Thr Lys Arg Ser Lys Tyr Leu Phe Leu Phe Leu Phe Leu Phe Phe 1 5 1015 Gly Ile Gln Thr Gly Ile Gln Ala Gln Leu Trp Ile Pro Pro Gly Arg 20 2530 Gln Tyr Met His Pro Thr Glu Pro Phe Thr Tyr Asp Leu Gly Ile Asn 35 4045 Lys Tyr Gln Lys Asp Tyr Tyr Leu Tyr Val Ala Pro Thr Val Asn Leu 50 5560 Asn Phe Gly Gly Asp Phe Gly Ala Ser Leu Thr Leu Pro Leu Asn Phe 65 7075 80 Leu Ile Tyr Asp Thr Glu Pro Lys Gln Glu Asn Ser Arg Ile Gly Lys 8590 95 Leu Arg Ser Phe Asp Tyr Asn Asp Lys Ser Asp Tyr Leu Arg Leu Ile100 105 110 Asn Asn Ile Trp Phe Gly Gln Tyr Gly Lys Tyr Thr Pro Gly GluIle 115 120 125 Thr Tyr Ser Ala Ser Leu Gly Lys Leu Phe Asp Gly Tyr IleGly His 130 135 140 Gly Thr Ile Val Asn Arg Tyr Val Asn Asn Gln Arg LeuAsp Val Tyr 145 150 155 160 Asn Val Gly Leu Gln Ala Asp Ile Asn Ser AspPhe Gly Gly Val Gln 165 170 175 Val Phe Ser Asn Ser Ile Tyr Thr Arg GluVal Ser Ser Ala Arg Val 180 185 190 Tyr Ile Arg Pro Phe Ala Val Gly TyrLys Leu Phe Asp Ile Val Thr 195 200 205 Gly Arg Ser Lys Phe Leu Thr MetMet Thr Ile Ala Gln Gly Asn Val 210 215 220 Ala Asp Glu Ala Gly Arg ArgLys Val Tyr Glu Glu Val Gly Ala Glu 225 230 235 240 Glu Lys Glu Ser TyrArg Ala Leu Ile Glu Asp Gln Lys Thr His His 245 250 255 Lys Lys Glu GluMet Ile Pro Val Asp Lys Lys Pro Glu Lys Pro Arg 260 265 270 Asn Leu LysGlu Ile Phe Asn Gln Asp Asn Trp Val Asn Arg Phe Ala 275 280 285 Ile GlyTyr Thr Thr Ala Phe Asp Thr Lys Ala Pro Ser Glu Leu Lys 290 295 300 PheAsp Thr Thr Gly Lys Leu Arg Val Asp Glu Asn Asp Asn Pro Leu 305 310 315320 Val Lys Ser Thr Glu Arg Leu Ser Ile Thr Gly Phe Asp Phe Glu Tyr 325330 335 Lys Leu Leu Ser Ala Lys Tyr Ile Glu Leu Thr Pro Tyr Tyr Asp Val340 345 350 Asn Lys Ile Lys Gln Ile Glu Asn Ala Lys Gly Thr His Tyr GlyAla 355 360 365 Ile Leu Arg Leu Gly Gly Lys Asp Ile Tyr Val Gln Ile LysPro Glu 370 375 380 Tyr Arg Asn Met Thr Ala Thr Tyr Ile Pro Met Tyr PheAsp Ser Phe 385 390 395 400 Tyr Glu Leu Glu Arg Phe Gln Ser Asn Leu GlnSer His Ile Pro Gln 405 410 415 Thr Lys Leu Glu Ala Pro Lys Leu Ala AspPro Asp Gly Ser Lys Ile 420 425 430 Lys Gly His Phe Thr Pro Val Leu PheAsn Phe Tyr Arg Phe Ala Ile 435 440 445 Glu Ser Asn Tyr Glu Asn Tyr SerGly Pro Asn Asn Ser Arg Val Phe 450 455 460 Leu Gly Val Tyr Ile Pro LeuGly Ser Met Phe Leu Ile Asn Gly Tyr 465 470 475 480 Tyr Met Lys Lys AlaPhe Lys Leu Asp Asp Arg Ser Gln Gly Ala Leu 485 490 495 Glu Leu Ala IleAsn Leu Gly Leu Val Thr Val Arg Leu Gln Asn Ile 500 505 510 Arg Lys TrpVal Tyr Asp Thr Ala Ser Ser Gln Tyr Glu Ala Gln Asp 515 520 525 Glu GlnLys Ile Leu Phe Ser Gly Gly Leu Tyr Phe 530 535 540 122 amino acidsamino acid single linear protein not provided TBP1 Protein 1..122 3 AspThr Ile Gln Val Lys Ala Lys Lys Asp Pro Gly Ile Ala Val Val 1 5 10 15Glu Gln Gly Arg Gly Ala Ser Ser Gly Tyr Ser Ile Arg Gly Met Asp 20 25 30Lys Asn Arg Val Ser Leu Thr Val Asp Gly Leu Ala Gln Ile Lys Ala 35 40 45Val Glu Ile Ser Lys Gly Ser Asn Ser Val Glu Gln Gly Ser Gly Ala 50 55 60Leu Ala Gly Ser Val Ala Phe Gln Thr Lys Ile Asp Pro Glu Lys Ser 65 70 7580 Phe Asn Lys Glu Ala Gly Ile Val Gln Ser Ala Arg Ile Thr Gly Ile 85 9095 Asn Leu Arg Ala Gly Val Tyr Asn Leu Leu Asn His Arg Tyr Gly Arg 100105 110 Asn Tyr Thr Phe Ser Leu Glu Met Lys Phe 115 120 122 amino acidsamino acid single linear protein not provided BtuB Protein 1..122 4 AspThr Leu Val Val Thr Ala Asn Arg Leu Pro Gly Val Asp Ile Thr 1 5 10 15Gln Asn Gly Gly Ser Gly Gln Leu Ser Ser Ile Phe Ile Arg Gly Thr 20 25 30Asn Ala Ser His Val Leu Val Leu Ile Asp Gly Val Arg Leu Asn Gln 35 40 45Arg Val Glu Tyr Ile Arg Gly Pro Arg Ser Ala Val Tyr Gly Ser Asp 50 55 60Ala Ile Gly Gly Val Val Asn Ile Ile Thr Thr Leu Asp Pro Glu Lys 65 70 7580 Ser Lys Gln Trp Glu Gly Ala Phe Gly Lys Ala Arg Ile Lys Gly Val 85 9095 Glu Val Arg Gly Lys Ile Ala Asn Leu Phe Asp Lys Asp Tyr Gly Arg 100105 110 Glu Tyr Thr Leu Ser Gly Ser Tyr Thr Phe 115 120 121 amino acidsamino acid single linear protein not provided Cir Protein 1..121 5 GluThr Met Val Val Thr Ala Ser Ser Val Pro Gly Val Gln Leu Thr 1 5 10 15Asn Glu Gly Asp Asn Arg Lys Gly Val Ser Ile Arg Gly Leu Asp Ser 20 25 30Ser Tyr Thr Leu Ile Leu Val Asp Gly Lys Arg Val Asn Glu Arg Ile 35 40 45Glu Val Val Arg Gly Pro Met Ser Ser Leu Tyr Gly Ser Asp Ala Leu 50 55 60Gly Gly Val Val Asn Ile Ile Thr Lys Leu Lys Pro Glu Thr Ser Glu 65 70 7580 Ser Trp Glu Leu Gly Leu Tyr Asn Lys Ala Arg Asn Gln Gly Val Glu 85 9095 Leu Arg Ala Gly Val Leu Asn Leu Gly Asp Lys Asp Leu Gly Arg Arg 100105 110 Tyr Phe Met Ala Val Asp Tyr Arg Phe 115 120 117 amino acidsamino acid single linear protein not provided IutA Protein 1..117 6 GluThr Phe Val Val Ser Ala Asn Arg Ile Pro Gly Leu Asp Val Ser 1 5 10 15Ser Arg Ser Arg Thr Asn Tyr Gly Met Asn Val Arg Gly Arg Pro Leu 20 25 30Val Val Leu Val Asp Gly Val Arg Leu Asn His His Ile Glu Val Ile 35 40 45Phe Gly Ala Thr Ser Leu Tyr Gly Gly Gly Ser Thr Gly Gly Leu Ile 50 55 60Asn Ile Val Thr Lys Leu Glu Gly Val Lys Val Asp Ser Tyr Glu Leu 65 70 7580 Gly Trp Arg Asp Lys Arg Arg Ile Tyr Gly Val Glu Leu Ser Phe Ser 85 9095 Ile Glu Asn Leu Phe Asp Arg Asp Tyr Arg Gly Arg Phe Gly Leu Asn 100105 110 Tyr Ser Val Leu Phe 115 125 amino acids amino acid single linearprotein not provided FhuA Protein 1..125 7 Asp Thr Ile Thr Val Thr AlaAla Pro Thr Pro Gly Val Ser Val Gly 1 5 10 15 Thr Arg Gly Ala Ser AsnThr Tyr Asp His Leu Ile Ile Arg Gly Phe 20 25 30 Ala Ala Glu Gly Gln SerGln Asn Asn Tyr Leu Asn Gly Leu Lys Leu 35 40 45 Gln Glu Arg Ala Glu IleMet Arg Gly Pro Val Ser Val Leu Tyr Gly 50 55 60 Lys Ser Ser Pro Gly GlyLeu Leu Asn Met Val Ser Lys Phe Ala Pro 65 70 75 80 Ser Lys Gly Lys GlnTyr Glu Val Gly Val Lys Gly Glu Ile Arg Ala 85 90 95 Arg Gly Val Glu ValAla Leu His Val Asn Asn Leu Phe Asp Arg Glu 100 105 110 Tyr Glu Arg GlnVal Val Ala Thr Ala Thr Phe Arg Phe 115 120 125 119 amino acids aminoacid single linear protein not provided PupA Protein 1 8 Asn Thr Val ThrVal Thr Ala Ser Ala Thr Pro Gly Ile Thr Met Ser 1 5 10 15 Gln Asp GlyGly Glu Arg Phe Asn Ile Tyr Ser Arg Gly Ser Ala Ile 20 25 30 Asn Ile TyrGln Phe Asp Gly Val Thr Thr Tyr Asp Arg Ile Glu Ile 35 40 45 Val Arg GlyAla Thr Gly Leu Met Thr Gly Ala Gly Asp Pro Ser Ala 50 55 60 Val Val AsnVal Ile Arg Lys Leu Asp Pro Glu Val Gly Lys Asn Tyr 65 70 75 80 Glu LeuGly Trp Lys Asp Gly Ala Glu Thr Lys Gly Val Asp Ala Thr 85 90 95 Leu AsnVal Asn Asn Ile Phe Asp Lys Lys Tyr Pro Arg Asn Ala Thr 100 105 110 ValThr Leu Arg Tyr Asp Phe 115 120 amino acids amino acid single linearprotein not provided IrgA Protein 1..120 9 Glu Thr Phe Val Val Ser AlaAsn Arg Val Pro Gly Val Thr Val Thr 1 5 10 15 Gly Gly Gly Asp Thr ThrAsp Ile Ser Ile Arg Gly Met Gly Ser Asn 20 25 30 Tyr Thr Leu Ile Leu ValAsp Gly Lys Arg Gln Thr Glu Arg Ile Glu 35 40 45 Val Ile Arg Gly Pro MetSer Thr Leu Tyr Gly Ser Asp Ala Ile Gly 50 55 60 Gly Val Ile Asn Ile IleThr Arg Leu Gln Pro Glu Thr Ser Ile Asn 65 70 75 80 Lys Glu Leu Ser LeuMet Asp Glu Ala Glu Thr Tyr Gly Ala Glu Ile 85 90 95 Lys Ala Ala Val TyrAsn Leu Phe Asp Gln Glu Val Gly Arg Arg Tyr 100 105 110 Trp Leu Gly LeuAsp Ile Ala Phe 115 120 124 amino acids amino acid single linear proteinnot provided FoxA Protein 1..124 10 Asp Thr Ile Glu Val Thr Ala Lys AlaThr Pro Gly Val Phe Thr Gly 1 5 10 15 Phe Ser Gly Gly Ala Thr Arg TyrAsp Thr Val Ala Leu Arg Gly Phe 20 25 30 His Gly Gly Asp Val Asn Asn ThrPhe Leu Asp Gly Leu Arg Leu Leu 35 40 45 Glu Arg Ile Asp Val Ile Lys GlyPro Ser Ser Ala Leu Tyr Gly Gln 50 55 60 Ser Ile Pro Gly Gly Val Val MetMet Thr Ser Lys Leu Lys Pro Met 65 70 75 80 Thr Ser Glu Gln Tyr Glu ValGly Ile Ile Gly Lys Val Asn Ser Gln 85 90 95 Gly Leu Glu Val Gln Leu AsnVal Asn Asn Ile Ala Asp Lys Lys Tyr 100 105 110 Glu Arg Ser Val Gln AlaThr Val Gly Tyr Asp Phe 115 120

What is claimed is:
 1. A purified monoclonal antibody which binds to anisolated protein having the amino acid sequence of SEQ ID NO:2.
 2. Apharmaceutical composition useful for ameliorating the onset of symptomscaused by pathogenic Leptospira in an animal comprising an effectiveamount of an antibody which binds OmpL2 in a pharmnaceuticallyacceptable carrier.
 3. The pharmaceutical according to claim 2, whereinthe effective amount of antibody is in the range of about 0.1 mg/kg ofbody weight to 2000 mg/kg of body weight.
 4. The pharmaceuticalaccording to claim 2, wherein the effective amount of antibody is in therange of about 0.1 mg/kg of body weight to 500 mg/kg of body weight. 5.The pharmaceutical according to claim 2, further comprising atherapeutic agent coupled to the antibody.
 6. The pharmaceuticalaccording to claim 5, wherein the therapeutic agent is a drug, aradioisotope, a lectin, or a toxin.